Molecular Dynamics Simulations of Native Protein Charging in Electrosprayed Droplets with Experimentally Relevant Compositions

Electrospray ionization-mass spectrometry (ESI-MS) has been widely used for the study of proteins given its preservation of much native-protein structure when transitioning to the gas-phase. Understanding the influence of experimental factors on ESI can provide insight into the correlation between protein structure and the resulting charge states, as well as the degree to which ‘native’ structure is maintained. Experimentally, it is challenging to characterize nanometer-scale electrosprayed droplets; however, molecular dynamics (MD) simulations pose an attractive solution by providing a molecular perspective of protein charging and transfer to the gas-phase during ESI. By resolving approximations used in past MD simulations of ESI, we demonstrate the capability of simulating electrosprayed droplets with experimentally relevant droplet composition and behavior. This is accomplished by modelling proton transfers between all titratable molecules in simulated droplets under atmospheric conditions; thus, enabling simulated droplets containing ammonium acetate that form experimentally observed protonated or deprotonated protein ions upon solvent evaporation. Application of the proposed protocol to several native proteins in positive- and negative-ion mode ESI produces charge-state distributions that are highly correlated to experimental mass spectra. Our simulations suggest that changes in residue basicity during the transition to the gas-phase play a significant role in moderating protein charging during native-ESI and can explain many experimentally observed trends. While applied towards native proteins in this work, novel insights into effects from the transition to gas-phase enable a deeper understanding of the ESI process itself and thus, are informative regardless of analyte.